1. Field of the Invention
The invention pertains to a composition designed for the detection of traces of human or animal blood, including therein a field kit and a procedure for the preparation of the composition in question. The invention further encompasses a procedure for the detection and the localisation of traces of human or animal blood.
The procedure of searching and the localisation of human and animal blood traces by means of chemiluminescence is an already well-established and documented factor.
The luminescence designates the ensemble of emission phenomena of electro-magnetic ultraviolet rays, either visible or in the infra-red range and that are not provoked by a thermal effect.
Certain molecules, the luminophores, possess the property, after they have been brought to an excited state, to emit this so-called <<cold light>> while in the process of returning to their fundamental state. The phenomenon may be provoked by different means and, depending on the nature of the energy source that brings a molecule to its excited state, different types of luminescence can be defined.
Chemiluminescence represents the emission of light produced directly or indirectly by a chemical reaction. In general, chemiluminescent reactions result from the process of oxidation.
For the detection of human or animal blood, the luminol (5-amino-2.3-dihydro-1.4-phthalazinedione) is the most commonly used chemiluminescent compound, although a number of other luminol compounds are equally employed.
In an aqueous solution, the chemiluminescent reaction of luminol requires the presence of a system of oxidisation and an alkaline environment (White E. H. & Roswell, D. F. 1985. Luminol chemiluminescence. Chemi-and Bioluminescence (Burr, J. G., ed.), Marcel Dekker, New York, pp. 215-244).
The oxidation of luminol leads to the formation of the ion aminophthalate in an excited state whose return to its fundamental state is accompanied by the emission of light.
The quantum efficiency yield of the reaction is feeble (approximately 0.01) and the emission spectrum presents a maximum at 430 nm (light-blue colour) (White, E. H. & Roswell, D. F. 1985. Luminol chemiluminescence. Chemi-and Bioluminescence (Burr, J. G., ed.), Marcel Dekker, New York, pp. 215-244).
The principal compounds that are able to act as catalysts in this light-emitting reaction are the transition metals (Cr3+, Mn4+, Fe2+, Fe3+, CO2+, Ni2+, Cu2+, Hg2+), free or complexed, hemin and peroxidase (enzyme extracted, for instance, from horse radish).
Hemin is a particular biochemical structure that forms an integral part of peroxidase. This structure is equally present in haemoglobin, which is the carrier protein of oxygen and of part of CO2 in the blood.
Hence, the presence of haemoglobin—thus, of blood—can be made evident by exploiting the ability of hemin to catalyse the chemiluminescence of luminol.
In other words, a mixture of luminol/oxidising agent/alkaline(base) agent, when coming into contact with blood, will emit light.
2. Discussion of Background Information
Already more than 60 years ago, a method to test for the presence of blood by means of this kind of compound was proposed as an empirical aid for detecting traces of blood as an instrument in criminal and forensic investigations.
Thus, in 1937, Specht, W. in The Chemiluminescence of hemin: an aid for finding and recognizing blood stains important for forensic purposes. Angewande Chemie, 10, 155-157, was the first to propose demonstrating at the scene of a crime the presence of blood to serve as evidence in criminal and forensic investigations, this by using a compound containing 0.1 part by weight of luminol, 3 parts by weight of an alkaline agent, being sodium carbonate, and 15 parts by weight of an oxidising agent, being hydrogen peroxide 30% diluted in 100 parts by weight of distilled water.
In 1937, Specht, W. in The Chemiluminescence of hemin: an aid for finding and recognizing blood stains important for forensic purposes. Angewande Chemie, 10, 155-157, studied and demonstrated the sensitivity of the luminescent reaction with this reactive agent on traces of dried blood, then diluted to 1:2 000 by volume.
In 1939 Proescher, F. & Moody, A. M. in Detection of blood by means of chemiluminescence. Journal of Laboratory Clinical Medicine., 24, 1183-1189, recalled that Specht, W., in 1937, in his article The Chemiluminescence of hemin: an aid for finding and recognizing blood stains important for forensic purposes. Angewande Chemie, 10, 155-157, had in tickets and brushwood conducted tests on blood exposed to the sun and to the rain and that, under these varying conditions, the chemiluminescent reaction had occurred.
As to their own efforts, they tested the compound of Specht, W., expounded in 1937 in The Chemiluminescence of hemin: an aid for finding and recognizing blood stains important for forensic purposes. Angewande Chemie, 10, 155-157, both on the blood from animals and humans up to dilutions of 1:1 000 000 and demonstrated thereby that the chemiluminescent reaction occurred on both occasions.
In 1951, Grodsky, M., Wright, K. & Kirk, P. L., in Simplified preliminary blood testing. An improved technique and comparative study of methods. Journal of the America Institute of Criminal Law and Criminology, 42, 95-104, noting that the sensitivity and the reactivity of luminol are very difficult to control while the hydrogen peroxide is being used in the capacity of oxidising agent, proposed the use of a perborate oxidising agent. He likewise proposed a field kit consisting of different receptacles each independently containing the said luminol, the perborate oxidising agent, and the alkaline agent sodium carbonate. This field kit would also hold accessories such as plastic and glass vaporisers, filter paper, a bottle of distilled water, a flashlight to allow for the mixing of the reactive agents in the dark, plus plastic receptacles to hold and carry along the traces of blood or other minute bits of evidence.
In that respect, Grodsky, M., Wright, K. & Kirk, P. L. in 1951, in the article Simplified preliminary blood testing. An improved technique and comparative study of methods. Journal of the America Institute of Criminal Law and Criminology, 42, 95-104, managed to optimise the chemiluminescent reaction of luminol, in the presence of very diluted blood, being a dilution of 1:5 000 000 by volume.
In 1939, Proescher, F. & Moody, A. M. in Detection of blood by means of chemiluminescence. Journal of Laboratory Clinical Medicine., 24, 1183-1189, just like Grodsky, M., Wright, K. & Kirk, P. L. in 1951 in Simplified preliminary blood testing. An improved technique and comparative study of methods. Journal of the America Institute of Criminal Law and Criminology, 42, 95-104, recommended to first spray the location with hydrochloric acid in order to decompose the haemoglobin and to improve the sensitivity level of the test.
In 1966, Weber, K. in Die Anweldung der Chemiluminescenz des Luminols in der Gerichtlichen Medizin und Toxicologie. I. Der Nachweis von Blutspuren. Deutsche Zeitschrift für die gesamte Gerichtliche Medizin, 57, 410-423, provided evidence of the fact that the use of a carbonate alkaline agent did merely provoke a slow reaction of haemoglobin oxidation and that, consequently, the luminescence was much weaker when such a carbonate alkaline agent was utilised. Therefore, he advocated the use of sodium hydroxide for an alkaline agent. In 1966, Weber, K. in Die Anweldung der Chemiluminescenz des Luminols in der Gerichtlichen Medizin und Toxicologie. I. Der Nachweis von Blutspuren. Deutsche Zeitschrift für die gesamte Gerichtliche Medizin, 57, 410-423, in his turn noted that the concentrations with luminol and hydrogen peroxide used in the reagents by Specht, W. in 1937 in The Chemiluminescence of hemin: an aid for finding and recognizing blood stains important for forensic purposes. Angewande Chemie, 10, 155-157, were too strong and provoked a concentration inhibition in the reaction of luminol linked to the concentration of the reagents and hence a disappearance of the chemiluminescence and a notable lowering in the evidence of blood traces. In 1966, Weber, K. in Die Anweldung der Chemiluminescenz des Luminols in der Gerichtlichen Medizin und Toxicologie. I. Der Nachweis von Blutspuren. Deutsche Zeitschrift für die gesamte Gerichtliche Medizin, 57, 410-423, thus proposed a composition consisting of 0.4 mmoles/l de luminol, 17.6 mmoles/l of hydrogen peroxide, and 45 mmoles/l of sodium hydroxide or potassium hydroxide, diluted in distilled water, for the detection of traces of dried or fresh blood up to a dilution ratio of 1:20 000 000.
In 1990, Grispino, R. R. J. in The effects of luminol on serological analysis of dried bloodstains Crime Laboratory Digest, 17 (1), 13-23, proposed using a luminol composition containing 5.6 mmoles/l of luminol, 472 mmoles/l of potassium or sodium carbonate and 100 mmoles/l of hydrogen peroxide H2O2, diluted in distilled water, for the detection of traces of blood dried during several days and of fresh blood in dilutions of this blood from 1:10 000 up to 1:100 000.
All of the above demonstrates that, for the purpose of detecting traces of blood at the scene of a crime, the use of a composition containing luminol in combination with an alkaline agent and an oxidising agent dates back a long way in the past.
With reference to this particular application, it is advocated to use hydrogen peroxide as an oxidising agent rather than sodium perborate, as sodium perborate is not readily soluble in water and impedes the vaporisation arrangement of the composition. Likewise, the use of sodium hydroxide or potassium hydroxide as an alkaline agent is advocated rather than a composition of carbonate, for the oxidation reaction of luminol was much slower with a carbonate compound than with a hydroxide one, meaning that the light emission was less intense with the former compound.
In 1966, Weber, K. in the article Die Anweldung der Chemiluminescenz des Luminols in der Gerichtlichen Medizin und Toxicologie. I. Der Nachweis von Blutspuren. Deutsche Zeitschrift für die gesamte Gerichtliche Medizin, 57, 410-423, thus proposed a composition of this type but containing rather weak quantities of luminol and of hydrogen peroxide, as too strong quantities of these compounds bring with them an inhibition in the luminol reaction and, consequently, a disappearance of the chemiluminescence and a notable lowering of the evidence of traces of blood.
Furthermore, Byrne, in the patents U.S. Pat. Nos. 5,770,116 and 5,833,887, has proposed the use of the chemiluminescent reaction of luminol in order to detect traces of blood loss in wounded game animals, on the hunting sites, in conditions of failing light and reduced visibility.
To this effect, Byrne, in the patents U.S. Pat. Nos. 5,770,116 and 5,833,887, advocates the use of a composition containing, in addition to luminol, sodium perborate for an oxidising agent and sodium carbonate as an alkaline agent.
Nevertheless, this composition presents difficulties linked to the utilisation of the sodium perborate and the sodium carbonate as already described supra.
This is particularly inhibiting for an application to be used on the occasion of the hunt, specifically when blood detection is sought not in the dead of (black) night but rather under the simple conditions of failing light, as is the case at the time of sundown, when the loss of luminous intensity emitted by the luminol reaction with the blood is particularly bothersome since this luminescent intensity at such a time may not be sufficiently noticeable for the hunter to detect the traces of blood left by his prey.
Moreover, all tests carried out in the past were conducted on much diluted blood samples.